Use of optical diffraction elements in detection methods

ABSTRACT

The invention relates to a method for determining luminescent molecules by means of optical excitation in confocal measurement volumes using a diffractive optical element. The method is particularly suitable for single-molecule determination, e.g. by means of fluorescence correlation spectroscopy or by means of dynamic light scattering. An apparatus suitable for carrying out the method according to the invention is furthermore disclosed.

[0001] The invention relates to a method for determining luminescentmolecules by means of optical excitation in confocal measurement volumesusing a diffractive optical element. The method is particularly suitablefor single-molecule determination, e.g. by means of fluorescencecorrelation spectroscopy or by means of dynamic light scattering. Anapparatus suitable for carrying out the method according to theinvention is furthermore disclosed.

[0002] The use of fluorescence correlation spectroscopy (FCS) for thedetection of analytes is known. EP-B-0 679 251 discloses methods andapparatuses for the detection of analytes by means of fluorescencespectroscopy, the determination being carried out in a convocalmeasurement volume which is part of the sample to be examined. However,parallel determination in a multiplicity of measurement volumes ispossible only to a limited extent and with high technical outlay.

[0003] One object on which the present application is based was toprovide methods and apparatuses for determining luminescent molecules,in particular by means of fluorescence correlation spectroscopy, whichpermits parallel determination in multiple confocal volume elements in asimple manner.

[0004] Consequently, the invention relates to a method for determiningluminescent molecules by means of optical excitation in confocalmeasurement volumes comprising the steps of:

[0005] (a) providing a sample comprising luminescent molecules,

[0006] (b) irradiating the sample with an optical excitation devicecomprising a light source, a diffractive optical element for splittinglight passing through into multiple foci and a focusing opticalarrangement for focusing multiple light beams passing through intomultiple confocal volume elements, and

[0007] (c) capturing emission radiation from the multiple confocalvolume elements.

[0008] Surprisingly, it has been found that splitting excitation lightradiated into the sample by means of diffractive optical elementspermits the production of multiple confocal volume elements whilstmaintaining a light intensity that is sufficient for detection purposes.This principle can be used for the production of multiple confocal pointfoci for the detection of individual molecules or a small number, e.g.up to 100 molecules in confocal volume elements with the aid of suitableexcitation or detection methods.

[0009] New microstructure fabrication technologies make it possible tofabricate optical elements with a previously calculatedthree-dimensional structure which represents a predetermined diffractiongrating for a light source. In this case, a desired point patterngenerated by light diffraction is converted by means of Fouriertransformation into a three-dimensional diffractive structure which issubsequently produced from a suitable material, for example by means ofphotolithographic etching.

[0010] Diffractive optical elements that may be used are, by way ofexample, three-dimensional optical gratings which, if appropriate, areapplied to an optically transparent carrier, and which diffract lightpassing through and generate a predetermined diffraction pattern, i.e. adesired arrangement of multiple optical foci, in the object plane bymeans of constructive and destructive interference. An essentialadvantage when using such diffractive optical elements is that arbitraryarrangements of the foci can be chosen by virtue of the form of theoptical grating. In this case, the multiple optical foci are preferablyformed by 1^(st)-order interferences, only minor light losses occurringas a result of 0th- or higher-order interferences.

[0011] The production of suitable diffractive optical elements isdescribed, for example in the Dissertation by F. Nikolaef at theChalmers Institute of Technologies (1999), in the Dissertation by M.Johansson at the Chalmers Institute of Technologies (2001) and in thepublication Johansson and Hard (Applied Optics 38(1999), 1302-1310).Suitable materials for producing the optical elements are plastics,glass and composites or other materials having optical transparency fora given wavelength which can be processed by means of photolithographicetching.

[0012] A preferred embodiment of the method according to the inventionrelates to the detection of luminescent molecules in the confocalmeasurement volumes by means of fluorescence correlation spectroscopy.The method may be carried out in principle according to the methoddescribed in EP-B-0 679 251. In this case, the measurement of one or afew analyte molecules is preferably effected in a measurement volume,the concentration of the analyte molecules to be determined preferablybeing ≦10⁻⁶ mol/l and the measurement volume preferably being ≦10⁻¹⁴ l.Substance-specific parameters are determined, which are determined bymeans of luminescent measurement at the analyte molecules. Theseparameters may be translation diffusion coefficients, rotation diffusioncoefficients or/and the excitation wavelength, the emission wavelengthor/and the lifetime of an excited state of a luminescent molecule or thecombination of one or more of these measurement quantities. Forspecifics about equipment details, reference is made to the disclosureof EP 0 679 251.

[0013] A preferred feature of the method according to the invention isthat the distance between the measurement volume in the sample liquidand the focusing optical arrangement of the light source is ≧1 mm,preferably 1.5 to 10 mm and particularly preferably 2 to 5 mm. It isfurthermore preferred for a gas phase region, which may contain air,protective gas or vacuum, to be arranged between the carrier containingthe sample liquid and the optical focusing device. Methods andapparatuses for carrying out FCS with a large distance between focusingoptical arrangement and confocal measurement volume are described in DE101 11 420.6.

[0014] The method according to the invention is suitable in principlefor carrying out any desired determination methods. A preferredembodiment relates to the determination of an analyte in a sample, e.g.for diagnostic applications or for screening for the purpose ofidentifying active substances which interact with a target substance.For this purpose, one or more analyte-binding substances which carry amarking group, in particular fluorescence marking group, that can bedetected by luminescence measurement are added to the sample. In thiscase, the method according to the invention preferably comprises adetermination of the binding of the marking substance to the analyte tobe detected. This detection may be effected for example by means of amobility change of the marking group on account of the binding to theanalyte or by means of a change in the luminescence of the marking group(intensity or/and decay time) on account of the binding to the analyte,or by means of so-called cross-correlation if a plurality of markinggroups are used.

[0015] The cross-correlation determination uses at least two differentmarkings, in particular fluorescence markings, whose correlated signalwithin the measurement volume is determined. This cross-correlationdetermination is described for example in Schwille et al. (Biophys. J.72 (1997), 1878-1886) and Rigler et al. (J. Biotechnol. 63 (1998),97-109).

[0016] The method according to the invention is suitable in particularfor the detection of biomolecules e.g. nucleic acids, proteins or otheranalyte molecules which occur in living organisms, in particular inmammals such as humans. Furthermore, it is also possible to detectanalytes which have been produced from biological samples in vitro, e.g.cDNA molecules which have been produced from mRNA by reversetranscription, or proteins which have been produced from mRNA or DNA byin vitro translation. The method is furthermore suitable for thedetection of analytes which are present as elements of a library and areintended to exhibit predetermined characteristics, e.g. binding to thedetection reagent. Examples of such libraries are phage libraries orribosomale libraries.

[0017] In a particularly preferred embodiment, the determinationcomprises a nucleic acid hybridization, one or more luminescence-markedprobes binding to a target nucleic acid as analyte. Such hybridizationmethods may be used for example for the analysis of gene expression,e.g. in order to determine a gene expression profile, or for theanalysis of mutations, e.g. single-nucleotide polymorphisms (SNP). Themethod according to the invention is also suitable, however, fordetermining enzymatic reactions or/and for determining nucleic acidamplifications, in particular in a thermocycling process. Preferredmethods for determining nucleic acid polymorphisms are described in DE100 56 226.4 and DE 100 65 631.5. A two-color or multicolorcross-correlation determination is particularly preferably carried outin this case.

[0018] In a further particularly preferred embodiment, the determinationcomprises the detection of a protein-protein or protein-ligandinteraction, in which case e.g. low-molecular-weight active substances,peptides, nucleic acids, etc. may be used as protein ligands. Atwo-color or multicolor correlation measurement is preferably carriedout for such determinations as well.

[0019] In an alternative preferred embodiment, so-called “molecularbeacon” probes or primers may be used, which—if they are present in thefree form—give rise to a different measurement signal in respect of theluminescence intensity or/and decay time than in the bound state.

[0020] A further preferred embodiment of the invention comprises amethod for the selection of particles in a substance library, a particlehaving a predetermined property being selected from a population,comprising a multiplicity of different particles. For this purpose,preferably, a population of different particles is provided, particleshaving a predetermined property are marked, the particles are conductedin a microchannel through a detection element, comprising multipleconfocal volume elements, in order to distinguish between marked andunmarked particles and marked particles are removed. The steps ofconduction and removal are preferably repeated at least once, theconcentration of the particles being reduced preferably by at least thefactor 10 ⁴ in a subsequent cycle compared with a preceding cycle. Theparticles may be selected for example from cells, parts of cellsurfaces, cell organells, viruses, nucleic acids, proteins andlow-molecular-weight substances. The method is also suitable for theselection of particles from the combinatorial library which may containgenetic packages such as phages, cells, spores or ribosomes. Theparticle population preferably contains more than 10⁶ and particularlypreferably more than 10¹⁰ different particles. The particles arepreferably marked with a luminescence marking group.

[0021] Yet another embodiment comprises carrying out a sequence analysisof polymers, in particular biopolymers, luminescent fragments of ananalyte present in the sample being determined. This embodiment issuitable in particular for carrying out a nucleic acid sequencing. Forthis purpose, a carrier particle with a nucleic acid moleculeimmobilized thereon is preferably provided, essentially all thenucleotide components of at least one base type in at least one strandof the nucleic acid molecule bearing a fluorescence marking. The carrierparticle is introduced into a sequencing apparatus comprising amicrochannel and retained there, e.g. by means of an IR capture laser,and individual nucleotide components are progressively cleaved from theimmobilized nucleic acid molecule, e.g. by treatment with anexonuclease. The cleaved nucleotide components are then conductedthrough a microchannel, preferably by means of a hydrodynamic flow, andthe base sequence of the nucleic acid molecule is determined there inconfocal volume elements on the basis of the sequence of cleavednucleotide components.

[0022] The method according to the invention enables a light beamoriginating from a light source, e.g. a laser, to be split into aplurality of optical foci. The light beam is preferably split into 2-32,in particular into 4-16, optical foci. By using a suitable focusingoptical arrangement, confocal volume elements are imaged in the samplefrom said optical foci. The confocal volume elements expediently have asize of 10⁻¹⁸ to 10⁻⁹ l, preferably of 10⁻¹⁸ to 10⁻¹² l and particularlypreferably of 10⁻¹⁶ to 10⁻¹⁴ l.

[0023] In order to capture radiation, in particular emission radiationfrom the multiple confocal volume elements, use is preferably made ineach case of a separate detector per volume unit or a spatiallyresolving detection matrix, e.g. an avalanche photodiode matrix or anelectronic detector matrix, e.g. a CCD camera.

[0024] Splitting the light beam into a plurality of optical foci permitsparallel determination in separate confocal volume elements. In apreferred embodiment, these confocal volume elements are provided inrespective separate containers of a carrier, preferably of amicrostructure.

[0025] The volume of these containers is preferably in the range of≧10⁻⁶ l and particularly preferably ≦10⁻⁸ l to 10⁻¹² l. Thus, thecarrier may comprise a microwave structure with a plurality ofdepressions for receiving sample liquid, which for example have adiameter of between 10 and 1000 μm. Suitable microstructures aredescribed e.g. in DE 100 23 421.6 and DE 100 65 632.3. Thesemicrostructures may be used for example for determining a nucleic acidhybridization in solution. The carrier furthermore preferably comprisesat least one temperature control element, e.g. a Peltier element, whichenables temperature regulation of the carrier or/and individual samplecontainers therein.

[0026] The carrier used for the method is expediently configured in sucha way that it enables optical detection of the sample. A carrier whichis optically transparent at least in the region of the sample containersis therefore preferably used. The carrier may in this case either befully optically transparent or contain an optically transparent base andan optically opaque covering layer with cutouts in the samplecontainers. Suitable materials for carriers are, for example, compositecarriers made of metals (e.g. silicon for the covering layer) and glass(for the base). Carriers of this type may be produced for example byapplying a metal layer with predetermined cutouts for the samplecontainers onto the glass. Plastic carriers, e.g. made of polystyrene orpolymers based on acrylate or methacrylate, may alternatively be used.It is furthermore preferred for the carrier to have a cover for thesample containers, in order to provide a system which is closed andessentially isolated from the surroundings during the measurement.

[0027] In a particularly preferred embodiment, a carrier is used whichcontains a lens element arranged in the beam path between measurementvolume and light source or detector of the optical apparatus. By way ofexample, the lens element may be fitted at the bottom of a microwavestructure. A lens element of the type may, for example, be produced byheating and shaping a photoresist using a master mold, e.g. made ofmetal such as silicon, and then applied onto the carrier. As analternative—e.g. when using carriers made of a fully plasticstructure—the lens elements may be integrated into the carrier, e.g.produced during production by injection molding. The numerical apertureof the optical measuring arrangement may be increased by using a lenselement, preferably a convex lens element. This numerical aperture ispreferably in the range of 0.5 to 1.2.

[0028] The carrier is furthermore preferably coated with a transparentantireflection coating in order to produce a higher refractive index. Byway of example, transparent oxides or nitrides may be used asantireflection coatings. Antireflection coatings are preferably alsoused on the optical arrangement.

[0029] Furthermore, electric fields may be generated in the carrier, inparticular in the region of the sample containers, in order to achieveconcentration of the analytes to be determined in the measurementvolume. Examples of electrodes which are suitable for generating suchelectric fields are described e.g. in DE 101 03 304.4.

[0030] The molecule to be determined may be bound to a carrierparticle—in particular in the case of a determination in the microwaveformat or in the case of single molecule sequencing. The carrierparticle has a size which enables movement in microchannels andretention at a desired position within a sequenzing apparatus. Theparticle size is preferably in the range of 0.5-10 μm and particularlypreferably 1-3 μm. Examples of suitable materials of carrier particlesare plastics such as polystyrene, glass, quartz, metals or semimetalssuch as silicon, metal oxides such as silicon dioxide or compositematerials which contain a plurality of the abovementioned components.Particular preference is given to using optically transparent carrierparticles, for example made of plastics, or particles having a plasticcore and a silicon dioxide shell.

[0031] Nucleic acid molecules are preferably immobilized on the carrierparticle via their 5′ end, e.g. by means of covalent or noncovalentinteraction. Polynucleotides are particular preferably bound to thecarrier by high-affinity interactions between the partners of a specificbinding pair, e.g. biotin/streptavidin or avidin, etc. As analternative, nucleic acid molecules may also be bound to the carrier bymeans of adsorption or covalently.

[0032] Carrier particles to which only a single nucleic acid molecule isbound are preferably used. Carrier particles of this type may beproduced by the nucleic acid molecules provided for the determinationbeing brought into contact with the carrier particles in a molar ratioof preferably 1:5 to 1:20, e.g. 1:10, under conditions under which thenucleic acid molecules are immobilized on the carrier.

[0033] The carrier-bound nucleic acid molecules, e.g. DNA molecules orRNA molecules, may be present in single-stranded form or double-strandedform. The nucleic acid molecules are preferably present insingle-stranded form. When used for sequencing, essentially all thenucleotide components, e.g. at least 90%, preferably at least 95%, ofall the nucleotide components, of at least one base type carry afluorescence marking group. It is also possible for essentially all thenucleotide components of at least two base types, for example 2, 3 or 4base types, to carry a fluorescence marking, each base type expedientlycontaining a different fluorescence marking group. Nucleic acids markedin this way may be produced by enzymatic primer extension on a nucleicacid matrix using a suitable polymerase, e.g. a thermostable DNApolymerase. A precise description of this method is found in DE 100 31840.1 and DE 100 65 626.9 and also the literature citations specifiedtherein.

[0034] The present invention relates still further to an apparatus fordetecting luminescent molecules, in particular for carrying out a methodas described above, comprising

[0035] (a) a carrier for receiving a sample which contains luminescentmolecules to be determined,

[0036] (b) an optical excitation device, comprising a light source, adiffractive optical element for splitting light passing through intomultiple foci and a focusing optical arrangement for focusing lightpassing through into multiple confocal volume elements for theexcitation of luminescence in the multiple confocal volume elements, and

[0037] (c) an optical detection device for detecting luminescence fromthe multiple confocal volume elements.

[0038] The carrier is preferably a microstructure with a plurality ofcontainers, preferably at least 10, particularly preferably at least 10²containers, for receiving a sample liquid, in which case the sampleliquid in the separate containers may originate from one or moresources. The introduction of the sample liquid to the containers of thecarrier may be effected e.g. by means of a piezoelectric liquid deliveryapparatus.

[0039] The containers of the carrier are configured in such a way thatthey enable binding of the detection reagent to the analyte in solution.The containers are preferably depressions in the carrier surface, inwhich case said depressions may in principle have any desired form, forexample circular, square, rhomboid, etc. The carrier may even comprise10³ or more separate containers.

[0040] As an alternative, the carrier may also contain a microchannelstructure with one or more microchannels which are suitable inparticular for a single-molecule sequencing method as described in DE100 31 840.1 and DE 100 65 626.9 or for a particle selection method asdescribed in DE 100 31 028.1.

[0041] The optical excitation device comprises a strongly focused lightsource, preferably a laser beam, which is focused onto the measurementvolume in the sample liquid by means of corresponding optical devices.The light source may also contain two or more laser beams, which arethen respectively focused onto the measurement volume by differentoptical arrangements before entering the sample liquid. If two or morelaser beams are used, a separate diffractive optical element may be usedfor each laser beam. The detection device may contain for example afiber-coupled avalanche photodiode detector or an electronic detector.However, it is also possible to use excitation or/and detection matricescomprising a point matrix of laser points produced by a diffractionoptical arrangement or a quantum well laser, as well as a detectormatrix produced by an avalanche photodiode matrix or an electronicdetector matrix, e.g. a CCD camera.

[0042] The carrier may be provided in prefabricated form, a plurality ofseparate containers of the carrier being filled with luminescence-markeddetection reagents, preferably luminescence-marked hybridization probesor primers. The carrier containing the detection reagents is thenexpediently dried.

[0043] In a preferred embodiment of the invention, a prefabricatedcarrier is provided which contains a multiplicity of separatecontainers, e.g. 100 containers, which are respectively filled withdifferent detection reagents, e.g. reagents for the detection of anucleic acid hybridization such as primers or/and probes. This carriermay then be filled with a sample originating from an organism to beexamined, e.g. a human patient, so that different analytes from a singlesample are determined in the respective containers. Carriers of thistype may be used for example to compile a gene expression profile, e.g.for the diagnosis of diseases, or for the determination of nucleic acidpolymorphisms, e.g. for the detection of a specific geneticpredisposition.

[0044] Finally, the invention relates to the use of a diffractiveoptical element for producing multiple optical foci for paralleldetermination of molecular interactions in multiple confocal volumeelements. Particular preference is attached to the use for fluorescencecorrelation spectroscopy, as explained in detail above. However, thediffractive optical elements may also be used for other methods, e.g.for dynamic laser light scattering, wherein the intensity fluctuation isdetermined by means of scattered light in confocal volume elements.

[0045] The invention will furthermore be explained by means of theaccompanying figures and examples.

[0046]FIG. 1 shows the diagrammatic illustration of an embodiment of themethod according to the invention. A light beam (2), e.g. a laser beam,is split into a plurality of partial beams (6), e.g. four partial beams(6), in a diffractive optical element (4) and conducted in an opticalfocusing device (8 a, 8 b). The partial beams (10) emerging there arebundled in such a way that they form confocal volume elements with apredetermined size at a predetermined distance from the optical focusingdevice.

[0047]FIG. 2 shows exemplary arrangements for multiple optical fociwhich can be produced by means of the diffractive optical element. Inprinciple, the arrangement of the foci is freely selectable. The fociare preferably formed from 1^(st)-order interferences. The losses due tointerferences of zeroth order and higher orders are low and preferablyamount to ≦30%.

[0048]FIG. 3 shows a further embodiment of the method according to theinvention. A laser beam (12) is split into a plurality of partial beams,e.g. four partial beams (16), by means of a diffractive optical element(14), which partial beams produce a predetermined pattern of opticalfoci. The partial beams are subsequently deflected by a dichroic mirror(18) and directed via a focusing optical arrangement (not shown) onto acarrier (20), where they form a plurality of confocal volume elements.Light (22) emitted from the confocal volume elements, e.g. byfluorescence, is captured and detected by detectors (24).

[0049]FIGS. 4A and 4B show preferred embodiments for suitable carriers.The carrier in accordance with FIG. 4A is a microstructure, e.g. in theform of a chip, having at least 10² separate sample containers (24). Thedistance between two sample containers is preferably 50-150 μm, e.g.100-110 μm. The density of the containers is preferably in the range of100-10,000 depressions per cm². The sample volume in the individualcontainers is preferably 10⁻⁶ to 10⁻¹² l. A confocal volume element formeasuring interactions between individual molecules or a small number ofmolecules is formed within the sample volume. The carrier shown in FIG.4A is suitable in particular for high-throughput diagnosis and activesubstance screening methods. If the number of containers within thecarrier is greater than the number of partial beams generated by thediffractive optical element, the carrier can be scanned in a pluralityof steps. For this purpose, the optical arrangement or/and the carriermay be readjusted in each case for the individual steps by means ofsuitable measures.

[0050]FIG. 4B shows a microchannel (26) with confocal volume elements(28) arranged therein. This microchannel structure may be used inparticular for single-molecule sequencing or for single-moleculeselection.

[0051]FIG. 5A shows a 2×2 pattern of multiple optical foci (brightspots), produced by a diffractive optical element.

[0052]FIG. 5B shows the autocorrelation curve of 10 fluorescencereporter molecules in one of the multiple foci. It was found that theautocorrelation curve is identical in each case in all four foci.

[0053]FIG. 5C shows a representation of a biochip microarray having adiameter of 300 μm with 25×25 depressions for receiving samples, whichis suitable for carrying out the method according to the invention.

1. A method for determining luminescent molecules by means of optical excitation in confocal measurement volumes comprising the steps of: (a) providing a sample comprising luminescent molecules, (b) irradiating the sample with an optical excitation device comprising a light source, a diffractive optical element for splitting light passing through into multiple foci and a focusing optical arrangement for focusing multiple light beams passing through into multiple confocal volume elements, and (c) capturing emission radiation from the multiple confocal volume elements.
 2. The method as claimed in claim 1, characterized in that the luminescent molecules are selected from luminescence-marked detection reagents which bind to an analyte present in the sample.
 3. The method as claimed in claim 1 or 2, characterized in that the determination comprises the measurement of a cross-correlated signal originating from a complex comprising analyte and detection reagent(s), said complex containing at least two different luminescence markings.
 4. The method as claimed in claim 1 or 2, characterized in that the determination comprises the measurement of a signal originating from a luminescence-marked detection reagent, the luminescence intensity or/and decay time of the detection reagent being different in the case of binding to the analyte than in the non-bound state.
 5. The method as claimed in claim 4, characterized in that the differences in the luminescence intensity or/and decay time are caused by quenching or energy transfer process.
 6. The method as claimed in one of the preceding claims, characterized in that the determination comprises a nucleic acid hybridization, one or more luminescence-marked probes binding to a target nucleic acid.
 7. The method as claimed in one of the preceding claims, characterized in that the determination comprises an enzymatic reaction.
 8. The method as claimed in one of the preceding claims, characterized in that the determination comprises a nucleic acid amplification, in particular a thermocycling process.
 9. The method as claimed in one of the preceding claims, characterized in that the determination comprises a mutation analysis for nucleic acids.
 10. The method as claimed in one of the preceding claims, characterized in that the determination comprises gene expression analysis for nucleic acids.
 11. The method as claimed in one of the preceding claims, characterized in that the determination comprises the measurement of a temperature-dependent melting curve during a nucleic acid hybridization.
 12. The method as claimed in one of the preceding claims, characterized in that the determination comprises a particle selection.
 13. The method as claimed in one of the preceding claims, characterized in that the determination comprises a nucleic acid sequencing.
 14. The method as claimed in one of the preceding claims, characterized in that a laser is used as light source.
 15. The method as claimed in one of the preceding claims, characterized in that the diffractive optical element used is a three-dimensional optical grating which, if appropriate, is applied on an optically transparent carrier, which diffracts light passing through and generates a predetermined diffraction pattern, comprising multiple optical foci.
 16. The method as claimed in one of the preceding claims, characterized in that the multiple optical foci are formed by 1^(st)-order interferences.
 17. The method as claimed in one of the preceding claims, characterized in that a light beam is split into 2 to 32, in particular 4 to 16, optical foci.
 18. The method as claimed in one of the preceding claims, characterized in that the confocal volume elements have a size of 10⁻¹⁸ to 10⁻⁹ l, preferably of 10⁻¹⁸ to 10⁻¹² l.
 19. The method as claimed in one of the preceding claims, characterized in that a separate detector or a spatially resolving detection matrix is in each case used for capturing emission radiation from the multiple confocal volume elements.
 20. The method as claimed in one of the preceding claims, characterized in that the distance between the focusing optical arrangement of the light source and a confocal volume element is ≧1 mm, preferably 1.5-10 mm and particularly preferably 2-5 mm.
 21. The method as claimed in one of the preceding claims, characterized in that the sample is thermally insulated from the light source and in particular from the focusing optical arrangement.
 22. The method as claimed in one of the preceding claims, characterized in that the sample is provided in a carrier with a plurality of separate containers, preferably at least 10² separate containers.
 23. The method as claimed in one of the preceding claims, characterized in that the optical measurement arrangement has a numerical aperture of 0.5 to 1.2.
 24. The method as claimed in one of the preceding claims, characterized in that the carrier contains a plurality of separate containers, preferably at least 10 separate containers, for receiving samples.
 25. The method as claimed in one of claims 1 to 21, characterized in that the sample is provided in a microchannel structure.
 26. The method as claimed in claim 25, characterized in that an analyte present in the sample is retained in the microchannel structure.
 27. The method as claimed in claim 25 or 26, characterized in that an analyte present in the sample is subjected to a separation reaction, fragments separated off from the analyte being determined.
 28. The method as claimed in one of claims 25 to 27, characterized in that the microchannel structure has one or more channels with a diameter of 1-100 mm.
 29. The method as claimed in one of the preceding claims, characterized in that an analyte present in the sample is coupled to a carrier particle.
 30. The method as claimed in claim 29, characterized in that a carrier particle made of plastic, glass quartz, metals, semimetals or made of a composite material is used.
 31. The method as claimed in claim 29 or 30, characterized in that the carrier particle has a diameter of 0.5-10 μm.
 32. An apparatus for determining luminescent molecules, in particular for carrying out a method as claimed in one of claims 1 to 31, comprising (a) a carrier for receiving a sample which contains luminescent molecules to be determined, (b) an optical excitation device, comprising a light source, a diffractive optical element for splitting light passing through into multiple foci and a focusing optical arrangement for focusing light passing through into multiple confocal volume elements for the excitation of luminescence in the multiple confocal volume elements, and (c) an optical detection device for detecting luminescence from the multiple confocal volume elements.
 33. The apparatus as claimed in claim 32, characterized in that the carrier comprises a microstructure with at least 10 separate containers for receiving samples.
 34. The use of a diffractive optical element for generating multiple optical foci for parallel determination of molecular interactions in multiple confocal volume elements.
 35. The use as claimed in claim 34 for fluorescence correlation spectroscopy.
 36. The use as claimed in claim 34 for dynamic laser light scattering. 